Biomedical subjects

P Calvo

Publications and source records attributed to P Calvo.

At least 55 records · Page 3Linked to original sources

PubMed1994

Precore mutant hepatitis B virus and outcome of chronic infection and hepatitis in hepatitis B e antigen-positive children.

Mutant hepatitis B virus (HBV), responsible for the lack of hepatitis B virus "e" antigen (HBeAg) secretion because of a translational stop codon at nucleotide 1896 of the HBV-DNA precore region (HBeAg minus HBV), has been detected worldwide in acute and chronic HBV infections and diseases. HBeAg minus HBV appears to condition the outcome of infection and to be involved in the pathogenesis of hepatitis B. We investigated the mutant prevalence and its clinical implications in 30 hepatitis B surface antigen/HBeAg-positive children (17 treated with interferon) with chronic hepatitis B. Wild-type and HBeAg minus HBV were characterized by quantitative oligohybridization assays in sera from 29 children followed up for a mean of 33 mo (12 mo to 9 y). At admission, 18 children (62%) circulated wild-type HBV alone; mutant HBV became detectable in two of them during the follow-up before HBeAg/anti-HBe seroconversion. Wild-type and HBeAg minus HBV were detected in 10 children (34.5%); mutant HBV levels were lower than 20% of total viremia in four of them and higher in six. Serum HBV-DNA from one child did not hybridize with our probes. HBeAg minus HBV was associated with older age (p < 0.009) and higher histologic activity (p < 0.069). HBeAg/anti-HBe seroconversion occurred independently from HBeAg minus HBV detection; it was observed in six (37.5%) of 16 children with wild-type HBV alone and in four (33.3%) of 12 children with mixed viremia.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1993

Differential expression of the alpha 1c adrenergic receptor subtype in rat tissues.

Three alpha 1 adrenergic receptor subtypes have been identified by molecular cloning techniques. However, the detection of the mRNA for the alpha 1c subtype has not yet been reported in rodent tissues. We have isolated from rat lung, by polymerase chain reaction techniques, a cDNA fragment corresponding to the third intracellular domain of the alpha 1c adrenergic receptor subtype. The nucleotide and deduced amino acid sequences of the cloned fragment presented an 87% and 96% identity, respectively, with those from the bovine receptor. Analysis of the distribution of the receptor mRNA in brain shows highest expression levels in cerebellum and striatum, whereas in non-neural tissues the receptor is mainly expressed in lung and heart. It was also found that the gene sequence which codes for this domain is not interrupted by introns.

Amino Acid Sequence↗

PubMed1993

Differential effect of chronic ethanol treatment on barbiturate and steroid modulation of muscimol-binding to rat brain cortex.

We report the differential alterations produced by chronic ethanol treatment on the modulation, by the barbiturate thiopental and the steroid 5 beta-pregnan-3 alpha-ol-20-one, of the binding of [3H]muscimol to membrane preparations from rat brain cortex. We found a clear barbiturate- and steroid-promoted enhancement of muscimol-binding to membranes in both control and ethanol-treated animals. However, the enhancements were higher in control animals, using the barbiturate, and in ethanol-treated rats, using the steroid, Bmax and Kd values were also differentially affected in control and ethanol-treated animals by the presence of the barbiturate or the steroid.

Animals↗

PubMed1993

Effect of bromoconduritol on glucosidase II from rat liver. A new kinetic model for the binding and hydrolysis of the substrate.

Bromoconduritol inhibits the p-nitrophenyl-glucosidase and maltase activities of glucosidase II purified from rat liver, an enzyme that removes the two alpha-1,3-linked glucose residues of the protein-bound oligosaccharide Glc2Man9GlcNAc2 in the processing of N-glycoproteins. The inactivation process exhibits pseudo-first-order kinetics. Previously, we have demonstrated the occurrence of two binding (active) sites in the glucosidase II for the substrates p-nitrophenyl alpha-D-glucopyranoside (pNphGlc) and maltose (high- and low-affinity sites). The inhibition kinetic studies with bromoconduritol indicate that the high- and low-affinity sites for pNphGlc correspond to high- and low-affinity sites for maltose, respectively. Bromoconduritol has no effect on the binding of the substrates (pNphGlc and maltose) to the high-affinity site, although it does modify the low-affinity site and hinders the binding of the two indicated substrates to this site. These results, together with previous reports, have prompted us to propose a new kinetic model of binding and hydrolysis of the physiological substrate of the enzyme, in which the outermost glucose residue would bind and be released at the high-affinity site, whereas the innermost glucose residue would do so at the low-affinity site.

Animals↗

PubMed1993

T-lymphocyte response to hepatitis C virus in different clinical courses of infection.

BACKGROUND: To assess the role played by the immune response in the outcome of hepatitis C virus infection, the CD4+ T-lymphocyte response to viral antigens was studied in infected individuals with different clinical courses. METHODS: Using six recombinant proteins of hepatitis C virus, the study assessed the proliferative responses of peripheral blood mononuclear cells from 41 patients with chronic hepatitis C, 11 patients whose chronic hepatitis was successfully treated with interferon alfa and 11 healthy HCV seropositive individuals. RESULTS: (1) Sixty-five percent of hepatitis C virus-seropositive individuals had CD4+ T-cell responses to viral proteins. (2) All viral proteins were immunogenic for T cells, although NS4 was the most immunogenic. (3) There was a significant correlation between the presence of CD4+ T cell responses to Core and a benign course of infection in healthy seropositives, most of whom were viremic. CONCLUSIONS: CD4+ T-cell responses to Core, although they do not coincide with virus clearance, are associated with a benign course of infection and may be required to maintain humoral and cellular responses protective against the disease.

Adolescent↗

PubMed1993

Hepatitis B virus unable to secrete e antigen and response to interferon in chronic hepatitis B.

BACKGROUND: Anti-hepatitis e antigen-positive chronic hepatitis B is a progressive liver disease associated with precore mutant hepatitis B virus (HBV) and poor response to interferon. Therefore, precore mutant HBV may behave as an interferon-resistant virus. The relations between the prevalences of wild-type and precore mutant HBVs in baseline viremias and response to interferon were analyzed. METHODS: Sera from 115 patients (59 treated and 56 untreated, followed up for 30 months) were tested using a quantitative oligonucleotide hybridization assay. RESULTS: Spontaneous or interferon-induced recoveries were observed in 28.5% (6 of 21) and 47.3% (18 of 38) or in 0% (0 of 35) and 19% (4 of 21) of the patients with wild-type prevalent or mutant prevalent HBVs, respectively. Relapses occurred in 85.7% (12 of 14) and 19.4% (4 of 21) of treated patients with prevalent precore mutant and prevalent wild-type HBV, respectively (P = 0.0001). High precore mutant HBV levels (> 20% of total viremia) were associated with the lack of permanent response to interferon (P = 0.01). CONCLUSIONS: Precore mutant HBV can influence the response to interferon when it reaches significant serum levels (> 20% of total viremia). Therefore, chronic hepatitis B should be treated as early as possible in its natural history before precore mutant HBV is selected as a prevalent virus.

Adolescent↗

PubMed1993

Characterization of the maltase activity of glucosidase II from rat liver. Kinetic model.

Glucosidase II is a key enzyme in the processing of N-glycoproteins since it removes the two glucose residues from the protein-linked oligosaccharide Glc2Man9GlcNAc2-R. We have studied the kinetics of the purified enzyme, using maltose as substrate. Analysis of data fitting to single and double-hyperbolic equations and the Eadie-Hofstee profile indicate that the enzyme has two binding (active) sites for the hydrolysis of maltose. The Km and Vmax values for the high-affinity site were 0.43 mM and 691 mU/mg, respectively, whereas the values for the low-affinity site were 57.7 mM and 2888 mU/mg, respectively. The Vmax/Km ratios were 1607 and 50.1 ml/min per g for the high- and low-affinity sites, respectively. A new kinetic model for this enzyme is proposed from the equilibria corresponding to the partial competitive inhibition produced by maltose on p-nitrophenyl-glucosidase activity. The amino acid composition of the enzyme has been established.

Amino Acids↗

PubMed1992

Autoradiographic study on [3H]flunitrazepam binding in rat cortex and hippocampus after chronic ethanol treatment.

Ethanol alters almost all membrane functions, but it behaves essentially like a benzodiazepine-type GABAergic agonist. The mechanism by which ethanol affects the GABA/benzodiazepine complex is not clear. We studied the possible changes in [3H]flunitrazepam binding induced by chronic ethanol treatment, using light microscopic autoradiography, to try to elucidate the controversy underlying this topic. This technique allows us to measure densities of benzodiazepine receptors in different anatomical brain areas--visual cortex and hippocampus--which seem to constitute the anatomical support for the behavioral and physiological responses affected by ethanol. Autoradiographic studies on the visual cortex and hippocampus from rats chronically treated with ethanol do not show statistically significant differences in the binding of [3H]flunitrazepam with respect to control animals. Furthermore, we did not find either rostro-caudal or medio-lateral differences in benzodiazepine receptor densities in each layer of the visual cortex.

Animals↗

PubMed1992

Effects of chronic treatment with ethanol and withdrawal of ethanol on levels of dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid in the striatum of the rat. Influence of benzodiazepines, barbiturate and somatostatin.

Administration of ethanol for 40 days, at 10.53 +/- 0.25 g/kg/day did not modify levels of dopamine (DA) or 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum of the rat; however, the concentration of homovanillic acid (HVA) and the ratio of turnover were increased in a statistically significant way (P < 0.05). Twenty-four hours after withdrawal of ethanol appears as the central time of the ethanol-induced abstinence syndrome, showing noticeable decreases in levels of DA (P < 0.05) and DOPAC (P < 0.05), with respect to control and chronically ethanol-treated groups. The concentrations of DA, DOPAC and HVA and ratio of turnover values showed a tendency to return to control normal levels of 48 hr after ethanol withdrawal, although the differences still showed statistical significance (P < 0.05). The intraperitoneal injection of saline, the water soluble benzodiazepine midazolam, the barbiturate thiopental and somatostatin, in single doses, resulted in a noticeable increase in levels of DA, DOPAC and HVA and ratio of turnover values. The intraperitoneal injection of midazolam produced statistically significant decreases in levels of DOPAC and ratio of turnover values (P < 0.01) in rats 48 hr after withdrawal of ethanol, with respect to control and chronically ethanol-treated animals, in contrast to the absence of changes produced when injecting thiopental or somatostatin.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1992

Effect of chronic ethanol treatment on the gamma-aminobutyric acid-mediated enhancement of [3H]flunitrazepam binding in rat cortex and hippocampus.

The mechanism by which ethanol affects the gamma-aminobutyric acid (GABA)/benzodiazepine complex is not clear. It is known that ethanol enhances the Cl- influx mediated by the GABAA receptor complex, and although chronic ethanol administration does not change the KD or Bmax for [3H]flunitrazepam binding, some reports have suggested that it could modify the modulation of benzodiazepine binding produced by GABA. In the present work, we studied the effect of chronic ethanol treatment on the modulation by GABA of [3H]flunitrazepam binding, using light microscopic autoradiography. This technique allows the measurement of densities of benzodiazepine receptors in different brain areas, the visual cortex and hippocampus, which appear to constitute the anatomical support for the behavioral and physiological responses affected by ethanol. We found enhancement of benzodiazepine binding by GABA at concentrations of greater than 10(-6) M for the various cortical and hippocampal areas studied from both control and ethanol-treated animals; this enhancement peaked at 10(-4) M GABA but decreased at 10(-3) M GABA. We found a clear effect of ethanol treatment on the modulatory properties of GABAA receptor, in both cortex and hippocampus, although only in cortex were the differences statistically significant between control and ethanol-treated animals.

Animals↗

PubMed1991

Glucosidase II from rat liver microsomes. Kinetic model for binding and hydrolysis.

Glucosidase II is an enzyme involved in glycoprotein biosynthesis, releasing both alpha-1,3-linked glucose residues from the protein-linked oligosaccharide Glc2Man9GlcNAc2-R in the processing of N-glycans. We studied the kinetic properties of the enzyme, purified to homogeneity and, for the first time, we have been able to demonstrate the occurrence of two active sites in this enzyme and to establish the mechanisms of binding and hydrolysis of the physiological substrate at its active site(s). The analyses of data fitting to single and double hyperbolic equations and the Eadie-Hofstee profile, together with the inhibition kinetics, demonstrate that the enzyme has two different active sites. The Km and Vmax. values for the high-affinity site (site 1) were 0.78 mM and 437 munits/mg respectively, whereas the values for the low-affinity site (site 2) were 481 mM and 13797 munits/mg respectively, for the p-nitrophenyl alpha-D-glucopyranoside substrate. The Vmax./Km ratios, which indicate the efficacy of an active site for a substrate, were 560 and 28.7 ml/min per g for active sites 1 and 2, respectively. The inhibition type, with respect to site 1, for glucose, maltose, D-glucone-delta-lactone, CaCl2 and MgCl2 was pure-competitive, partial-competitive, parabolic, non-competitive and non-competitive respectively. Ki values for glucose, maltose, CaCl2 and MgCl2 were 6.75, 2.05, 10.60 and 14.20 mM respectively. Thus glucose would bind to active site 1, maltose to site 2 (and near to site 1) and D-glucone-delta-lactone to either site 1 or 2. The following hydrolysis mechanism for the physiological substrate (Glc2Man9GlcNAc2-protein) of glucosidase II may be concluded from all the foregoing kinetic evidence: the external glucose would be the first released residue, at active site 2, thereafter producing Glc1Man9GlcNAc2-protein; the remaining glucose would be released at active site 1, delivering the Man9GlcNAc2-protein product, which would leave the enzyme.

Animals↗

PubMed1991

Treatment with interferon of chronic hepatitis B associated with antibody to hepatitis B e antigen.

Persistence of HBV replication (serum HBV-DNA and intrahepatic HBcAg) and markers of HBV-induced (IgM anti-HBc positive) liver disease in anti-HBe-positive patients characterize a peculiar form of chronic hepatitis B. This form of hepatitis B prevails in the Mediterranean Basin, Middle and Far East and is associated with the infection of an HBV variant that lacks the capacity to produce HBeAg. We analysed the results of interferon treatment of 90 patients with chronic anti-HBe-positive hepatitis included in four randomized controlled trials. Interferon inhibited viral replication to undetectable levels and ALT normalized in about 70% of patients. However, the effect was transient in the majority of cases and hepatitis B relapsed in 41 to 90% of patients. A discrepancy in the rate of relapses could be explained by a significant difference in patients populations with a higher prevalence of cirrhotic patients in studies with poorer response. Therefore, in advanced anti-HBe-positive chronic hepatitis B, interferon shows a lower efficacy than in HBeAg-positive patients. The earlier treatment starts, the more efficacious is the response to interferon. Future clinical trials should focus on higher doses for longer periods, repeated courses or on combination therapy with nucleoside analogs or immuno-stimulant drugs.

Chronic Disease↗

PubMed1991

Purification and characterization of different "acid" beta-galactosidases from sheep kidney.

1. Two "acid" forms, Am and Al, of beta-galactosidase from sheep kidney have been isolated and purified 349- and 154-fold, respectively, with a recovery of about 8%. 2. Their mol. wts were about 450,000 and 230,000, respectively. Am seems to be a dimer of Al. The aggregation is stimulated by NaCl. 3. The "acid" beta-galactosidase has a pH optimum between 4.0 and 5.0 for both forms. They are located in the lysosomes. The optimal temperature is 37 degrees C and 40 degrees C for Al and Am forms, respectively. 4. Three peaks were detected by isoelectric focusing. After sialidase treatment, these peaks were obtained at higher pH values. 5. The activation energy values were 10.75 and 11.72 kcal/mol for Am and Al, respectively. 6. A variety of chemicals were tested as possible activators or inhibitors. The enzyme is strongly inhibited by gamma-D-galactonolactone, and the kinetic evidence suggests a competitive inhibition in all cases.

Animals↗

PubMed1991

[3H]-flunitrazepam binding after morphine treatment and under abstinence syndrome.

Chronic morphine treatment produced increases in [3H]-flunitrazepam binding in some hippocampal areas of the rat brain. The differences in binding were statistically significant in some cases. Both morphine-dependent and morphine-deprived (abstinence syndrome) animals showed an identical response in binding, which confirms a real, although small, increase in benzodiazepine binding sites in the hippocampus after morphine treatment, that is not affected by a naloxone-induced abstinence syndrome under the conditions studied. These findings support the hypothesis of a morphine-induced up-regulation of benzodiazepine binding sites in the hippocampus. A possible different response in benzodiazepine binding sites 1 and 2 could explain the different findings reported in the literature. Our data suggest that the detected increase in benzodiazepine binding would be mainly due to type 2 binding sites, since the hippocampus has a higher density of this type of benzodiazepine binding sites.

Animals↗

PubMed1991

Improvement of ocular penetration of amikacin sulphate by association to poly(butylcyanoacrylate) nanoparticles.

The main objective of this paper was to investigate the ability of polycyanoacrylate nanoparticles to improve the corneal penetration of hydrophilic drugs. Three different nanoparticle formulations were prepared by changing the nature of the stabilizer agent (Dextran 70000, Synperonic F 68 and sodium lauryl sulphate). The significant influence of the stabilizer type on the particle size, electrophoretic mobility and on the drug loading efficiency was proved. Moreover, the ocular disposition of amikacin was affected by its association to nanoparticles, displaying the most interesting results when Dextran 70000 was employed for preparation of nanoparticles. The increase of the amikacin concentration in cornea and aqueous humour was statistically significant for this nanoparticle formulation with respect to the other formulations and the control solution. The in-vitro release profiles obtained using a dialysis system were similar for all the nanoparticle formulations and for the control solution, indicating that drug molecules are desorbed from the nanoparticles quickly enough to maintain the equilibrium concentration in the dialysis system.

Administration, Topical↗

PubMed1991

Glucosidase II from control and ethanol-treated rats. Purification and properties.

Liver glucosidase II from control and ethanol-treated rats was purified and its physical and catalytic properties studied. No significant variation was found in the purification and properties of the enzyme from either source (ethanol-treated and control rats), except for activity after storage. Glucosidase II was purified to electrophoretic homogeneity from liver microsomes by solubilization, protamine sulphate precipitation, and anion exchange (DEAE-Sephacel) and affinity (Con A-Sepharose-4B) chromatographies. The native enzyme molecule is a tetramer (Mr 425 +/- 10 kDa) with identical subunits (Mr 106 +/- 3 kDa). Km values, determined at pH 6.8 for the p-nitrophenyl-glucosidase activity of glucosidase II from control and ethanol-treated rats, were 1.20 +/- 0.12 and 1.14 +/- 0.13 mM, respectively. The Arrhenius plot was linear, and the value for the apparent activation energy, calculated from this plot, was 56.64 kJ/mol. The p-nitrophenyl-glucosidase activity of glucosidase II from control and ethanol-treated rats was inhibited to the same extent by NH4+, by the divalent cations Ca2+ and Mg2+, and by methanol, ethanol, 2-propanol, n-propanol, isobutanol and n-butanol.

Alcoholism↗

PubMed1990

Autoradiographic localization of alpha 2-adrenoceptors in chick brain.

alpha 2-Adrenoceptors were localized in the chick brain by 'in vitro' receptor autoradiography using [3H]UK 14304 as a ligand. High or very high densities of binding sites were found in the hyperstriatum, tuberculum olfactorium, hypothalamic nuclei, tectum opticum and some medullary nuclei. Comparatively, intermediate densities were observed over the thalamic nuclei and locus ceruleus, among others. Low densities of alpha 2-adrenoceptors were detected in the paleostriatum, hippocampus and cerebellum. Our data indicate that alpha 2-adrenoceptors in the chick brain present similar properties and homologous anatomical distribution to those reported in mammalian brain.

Adrenergic alpha-Agonists↗

PubMed1989

Purification and characterization of a broad specificity beta-glucosidase from sheep liver.

1. A soluble beta-glucosidase from sheep liver has been isolated and purified 114-fold by conventional enzyme fractionation procedures. The specific activity of the purified enzyme was 5910 mU/mg of protein. 2. The enzyme has a broad specificity and hydrolyzes the p-nitrophenyl derivatives of beta-D-glucose, beta-D-galactose, beta-D-fucose, beta-D-xylose and alpha-L-arabinose. The best Vmax/Km value corresponds to the beta-glucosidase activity. 3. The enzyme has a pH optimum between 4.5-5.5 for all the activities, and mol. wt 95,000. 4. A variety of chemicals was tested as possible activators or inhibitors. 5. The enzyme is strongly inhibited by aldono 1-5 lactones and DMDP. 6. The kinetic evidences suggest a substrate activation model and the existence of two active sites (a "gluco-fuco" site and a "galacto" site). 7. The activation energies were calculated from beta-glucosidase and beta-galactosidase activities.

Alkaloids↗